U.S. patent application number 10/748094 was filed with the patent office on 2005-06-30 for non-pegylated long-circulating liposomes.
This patent application is currently assigned to Bharats Serums & Vaccines LTd.. Invention is credited to Daftary, Gautam Vinod, Pai, Srikanth Annappa, Rivankar, Sangeeta Hanurmesh.
Application Number | 20050142178 10/748094 |
Document ID | / |
Family ID | 52666552 |
Filed Date | 2005-06-30 |
United States Patent
Application |
20050142178 |
Kind Code |
A1 |
Daftary, Gautam Vinod ; et
al. |
June 30, 2005 |
Non-pegylated long-circulating liposomes
Abstract
The present invention provides a long circulating non-pegylated
liposomal doxorubicin hydrochloride composition for parenteral
administration and a process for its preparation. The circulation
time in Swiss albino mice is at least 25 times longer than
conventional non-liposomal formulations. The non-pegylated
liposomes are stable, exhibit low toxicity and have been found to
be efficacious in different tumor models.
Inventors: |
Daftary, Gautam Vinod;
(Thane, IN) ; Pai, Srikanth Annappa; (Thane,
IN) ; Rivankar, Sangeeta Hanurmesh; (Thane,
IN) |
Correspondence
Address: |
Teresa A. Lavenue
Kenyon & Kenyon
Suite 700
1500 K. Street, N.W.
Washington
DC
20005
US
|
Assignee: |
Bharats Serums & Vaccines
LTd.
Thane
IN
|
Family ID: |
52666552 |
Appl. No.: |
10/748094 |
Filed: |
December 31, 2003 |
Current U.S.
Class: |
424/450 ;
514/34 |
Current CPC
Class: |
A61K 31/704 20130101;
A61P 35/00 20180101; A61K 9/127 20130101 |
Class at
Publication: |
424/450 ;
514/034 |
International
Class: |
A61K 009/127; A61K
031/704 |
Claims
1. A process for manufacture of long circulating non-pegylated
liposomes comprising; forming a lipid film by evaporating a solvent
from a lipid solution comprising one or more phospholipids, a
sterol and a solvent; hydrating the lipid film with an aqueous
hydration media to form non pegylated liposomes; wherein the amount
of aqueous hydration media used is in the range of 10 to 35 ml for
each mmole of phospholipid present in the lipid solution.
2. The process of claim 1 wherein the amount of aqueous hydration
media used is 30 ml for each mmole of phospholipid in the lipid
solution.
3. The process of manufacture of non-pegylated liposomes of claim 1
further comprising loading the liposomes with a therapeutic or
diagnostic agent.
4. The process of claim 3, wherein the therapeutic agent is an
antineoplastic agent.
5. The process of claim 4, wherein the antineoplastic agent is
selected from the group consisting of Doxorubicin hydrochloride,
Daunorubicin hydrochloride, and Epirubicin hydrochloride.
6. The process of claim 5, wherein the antineoplastic agent is
Doxorubicin hydrochloride.
7. The process of claim 1, wherein the molar ratio of phospholipid
to sterol is from about 1:0.1-1:2.
8. The process of claim 7, wherein the wherein the molar ratio of
phospholipid to sterol is from about 1:0.7.
9. The process of claim 1, wherein the aqueous hydration media
comprises ammonium sulfate and sucrose.
10. The process of claim 9, wherein the concentration of ammonium
sulfate in aqueous hydration media is not less than 125
mmoles/liter.
11. The process of claim 1, wherein the phospholipid has a phase
transition temperature of 40.degree. C. to 60.degree. C.
12. The process of claim 11, wherein the phospholipid has a minimum
of sixteen carbons fatty acid chain.
13. The process of claim 12, wherein the phospholipid is selected
from the group consisting of Distearoyl phosphatidylcholine (DSPC),
Dipalmitoyl phosphatidylcholine (DPPC), Hydrogenated soya
phosphatidylcholine (HSPC) and derivatives of such
phospholipids.
14. The process of claim 13, wherein the phospholipid is distearoyl
phosphatidylcholine (DSPC) and wherein the sterol is
cholesterol.
15. The process of claim 1, wherein the non-pegylated liposomes are
successively extruded through series of filters having pore sizes
from 0.4 .mu.m to 0.05 .mu.m for sizing.
16. A liposome manufactured by the process of claim 1.
17. The liposome of claim 16, wherein the phospholipid comprises
distearoyl phosphatidylcholine (DSPC) and the sterol comprises
cholesterol.
18. The liposome of claim 16, wherein the non-pegylated liposome
further comprises a therapeutic or diagnostic agent.
19. The liposome of claim 18, wherein said therapeutic agent
comprises an antineoplastic agent.
20. The liposome of claim 19, wherein the antineoplastic agent is
selected from the group consisting of Doxorubicin hydrochloride,
Daunorubicin hydrochloride, and Epirubicin hydrochloride.
21. The liposome of claim 20, wherein the antineoplastic agent is
Doxorubicin hydrochloride.
22. The liposome of claim 16, wherein the average size of liposome
is 0.06 .mu.m to 0.16 .mu.m in diameter.
23. A long circulating non-pegylated liposomal doxorubicin
composition for parenteral administration comprising, doxorubicin
hydrochloride non-pegylated liposomes, histidine hydrochloride, and
sucrose; wherein the doxorubicin non-pegylated liposomes comprise
distearoylphosphatidyl choline, cholesterol, sucrose; wherein the
liposomes have an average size 0.06 .mu.m to 0.16 .mu.m; and
wherein the non-pegylated doxorubicin liposomes have a circulation
time in blood at least 25 times longer than that of ADRIAMYCIN when
tested in Swiss albino mice at equivalent doses.
24. The composition of claim 23, wherein the doxorubicin
concentration encapsulated in the liposomes is from 1mM to 10mM
expressed as doxorubicin hydrochloride.
25. The composition of claim 24, wherein the doxorubicin
hydrochloride concentration is from 3mM to 7mM.
26. The composition of claim 25, wherein the doxorubicin
hydrochloride concentration is about 3.45mM.
27. The composition of claim 25, wherein the doxorubicin
hydrochloride concentration is about 6.9mM.
28. The composition of claim 23, wherein the molar ratio of
distearoylphosphatidyl choline to cholesterol is from 1:0.6 to
1:0.8.
29. The composition of claim 28, wherein the molar ratio of
distearoylphosphatidyl choline to cholesterol is about 1:0.7.
30. The composition of claim 23, wherein the molar ratio of
doxorubicin hydrochloride to distearoylphosphatidyl choline is from
1:2 to 1:15.
31. The composition of claim 30, wherein the molar ratio of
doxorubicin hydrochloride to distearoylphosphatidyl choline is
about 1:3.5.
32. The composition of claim 23, wherein the sucrose concentration
is from 0.1M to 0.5M.
33. The composition of claim 32, wherein the sucrose concentration
is about 0.29 M.
34. The composition of claim 23, wherein the concentration of
histidine hydrochloride is from 1 to 100mM.
35. The composition of claim 34, wherein the concentration of
histidine hydrochloride is from 8 to 12mM.
36. The composition of claim 35, wherein the concentration of
histidine hydrochloride is about 10 mM.
37. The composition of claim 23, wherein the average size of the
liposomes is from 0.08 .mu.m to 0.12 .mu.m.
38. The composition of claim 23, wherein the doxorubicin
hydrochloride is present at 2 mg/ml; and wherein the molar ratio of
doxorubicin to DSPC is 1:3.5; and wherein the ratio of DSPC to
cholesterol is 1:0.7.
39. The composition of claim 23, wherein the doxorubicin
hydrochloride is present at 4 mg/ml; and wherein the molar ratio of
doxorubicin to DSPC is 1:3.5; and wherein the ratio of DSPC to
cholesterol is 1:0.7.
40. The composition of claim 23, wherein circulation time (t1/2) in
blood is at least 40 times longer than that obtained with
ADRIAMYCIN when tested in Swiss albino mice at equivalent
doses.
41. A method for reducing tumor growth comprising administering the
composition of claim 23.
42. A method for reducing tumor growth comprising administering the
composition of claim 38 and 39.
43. A process for manufacture of a long circulating non-pegylated
liposomal doxorubicin composition for parenteral administration
comprising (j) dissolving lipids comprising
Distearoylphosphatidylcholine (DSPC) and cholesterol in a single
solvent or in a mixture of solvents, (k) removing said solvents
before or after hydrating the lipids by addition of an aqueous
hydration media to form liposomes in a liposomal composition,
wherein said aqueous hydration media comprises ammonium sulfate and
sucrose, and wherein the aqueous hydration media is added in
quantities in the range of 10 ml to 35 ml per each mmole of DSPC;
(l) sizing the liposomes in the liposomal composition obtained at
the end of step (b), to about 0.060 .mu.m-0.16 .mu.m; (m) removing
extraliposomal ammonium sulfate from the liposomal composition that
has undergone sizing at step (c), using a sucrose-histidine buffer
solution comprising histidine hydrochloride and sucrose; (n)
dissolving doxorubicin hydrochloride in said sucrose-histidine
buffer solution to obtain a solution of at least 25mM doxorubicin
hydrochloride concentration; (o) admixing doxorubicin hydrochloride
solution obtained at step (e) and the liposomal composition
obtained at the end of step (d) to obtain doxorubicin hydrochloride
loaded liposomal composition; (p) removing extraliposomal
doxorubicin hydrochloride from the liposomal composition by a
process selected from the group consisting of tangential flow
filtration, column chromatography and treatment with resins; (q)
making up the volume of the liposomal composition obtained at the
end of step (g) with said sucrose-histidine buffer solution to
obtain a liposomal composition of a desired concentration of
doxorubicin hydrochloride; (r) filtering aseptically, the liposomal
composition through a sterile 0.2.mu. sterilising grade filter into
a sterile container to obtain said liposomal doxorubicin
composition.
44. A process for manufacture of a long circulating non-pegylated
liposomal doxorubicin composition for parenteral administration as
claimed in claim 43 further comprising, filling the liposomal
doxorubicin hydrochloride composition into sterile depyrogenated
containers and sealing the container under cover of an inert
gas.
45. A process for manufacture of a long circulating non-pegylated
liposomal doxorubicin composition for parenteral administration as
claimed in claim 43 wherein the concentration of ammonium sulfate
in the aqueous hydration media is not less than 125mM per
liter.
46. A process for manufacture of a long circulating non-pegylated
liposomal doxorubicin composition for parenteral administration as
claimed in claim 43, wherein, the molar ratio of sucrose to
histidine hydrochloride in the sucrose-histidine buffer solution
used in step d) is between 29:0.1 to 29:10.
47. A process for manufacture of a long circulating non-pegylated
liposomal doxorubicin composition for parenteral administration as
claimed in claim 46, wherein, the molar ratio of sucrose to
histidine hydrochloride in the sucrose-histidine buffer solution is
29:1.
48. A process for manufacture of a long circulating non-pegylated
liposomal doxorubicin composition for parenteral administration as
claimed in claim 43, wherein the doxorubicin hydrochloride
concentration is from 1mM to 10mM.
49. A process for manufacture of a long circulating non-pegylated
liposomal doxorubicin composition for parenteral administration as
claimed in claim 48, wherein the doxorubicin hydrochloride
concentration is about 3.45mM.
50. A process for manufacture of a long circulating non-pegylated
liposomal doxorubicin composition for parenteral administration as
claimed in claim 43, wherein the molar ratio of
distearoylphosphatidyl choline:cholesterol is from 1:0.6 to
1:0.8.
51. A process for manufacture of a long circulating non-pegylated
liposomal doxorubicin composition for parenteral administration as
claimed in claim 50, wherein the molar ratio of
distearoylphosphatidyl choline:cholesterol is about 1:0.7
52. A process for manufacture of a long circulating non-pegylated
liposomal doxorubicin composition for parenteral administration as
claimed in claim 43, wherein the molar ratio of doxorubicin
hydrochloride:distearoylphosphatidyl choline is from 1:2 to
1:15.
53. A process for manufacture of a long circulating non-pegylated
liposomal doxorubicin composition for parenteral administration as
claimed in claim 52, wherein the molar ratio of doxorubicin
hydrochloride:distearoylphosphatidyl choline is about 1:3.5
54. A process for manufacture of a long circulating non-pegylated
liposomal doxorubicin composition for parenteral administration as
claimed in any claim 43, wherein the sucrose concentration is from
0.1 M to 0.5M.
55. A process for manufacture of a long circulating non-pegylated
liposomal doxorubicin composition for parenteral administration as
claimed in any claim 54, wherein the sucrose concentration is from
0.25M to 0.3M.
56. A process for manufacture of a long circulating non-pegylated
liposomal doxorubicin composition for parenteral administration as
claimed in any claim 55, wherein the concentration of histidine
hydrochloride is from 1mM to 100mM.
57. A process for manufacture of a long circulating non-pegylated
liposomal doxorubicin composition for parenteral administration as
claimed in claim 55, wherein the concentration of histidine
hydrochloride is from 8mM to 12mM
58. A process for manufacture of a long circulating non-pegylated
liposomal doxorubicin composition for parenteral administration as
claimed in claim 56, wherein the concentration of histidine
hydrochloride is about 10mM.
59. A process for manufacture of a long circulating non-pegylated
liposomal doxorubicin composition for parenteral administration as
claimed in claim 43, wherein half circulation time (t.sub.1/2) in
blood is at least 25 times longer than that obtained with
ADRIAMYCIN when tested in Swiss albino mice at equivalent doses
60. A process for manufacture of long circulating non-pegylated
sized liposomes comprising; dissolving one or more phospholipids,
and a sterol in a solvent or mixture of solvents; removing said
solvents before after hydrating the phospholipids by addition of a
aqueous hydration media to form non-pegylated liposomes; wherein
the amount of the aqueous hydration media used is in the range of
10 to 35 ml for each mmole of phospholipid present in the lipid
solution; sizing the non-pegylated liposomes to about 0.06 .mu.m to
0.1 .mu.m to form a liposomal composition; removing extra-liposomal
hydration salt from the liposomal composition using
sucrose-histidine buffer solution to form non-pegylated sized
liposomes.
Description
[0001] This application claims priority to provisional application
1101/Mum/02, filed on Dec. 31, 2002, and Indian application ______
, filed on Dec. 31, 2003 and PCT application ______ filed on Dec.
31, 2003, all of which are hereby incorporated by reference in
their entirety.
FIELD OF THE INVENTION
[0002] The present invention relates to non-pegylated, long
circulating liposomes for parenteral administration and the
manufacture thereof, that can be used to contain and deliver
diagnostic or therapeutic agents.
BACKGROUND OF THE INVENTION
[0003] Liposomes are commonly composed of phospholipid and/or
sterols and consist of a vesicular structure based on lipid
bilayers surrounding aqueous compartments. They vary widely in
their physicochemical properties such as size, surface charge, and
phospholipid composition.
[0004] Liposomes have received increasing attention as possible
carriers for diagnostic or therapeutic agents. For example,
liposomes have been used to deliver diagnostic agents such as
contrast agents for magnetic imaging such as
Gd:diethylenetriaminepentacedic acid chelate (Gd-DTPA) (See e.g.
U.S. Pat. No. 6,132,763) and therapeutic agents such as
anthracycline agents, which have been shown to exhibit marked
activity against a wide variety of neoplasms. (See e.g. U.S. Pat.
No. 4,769,250).
[0005] However, liposomes cause aggregation in the blood by their
mutual reaction with various blood plasma proteins and are captured
by the reticuloendothelial system (RES). For example, Kupfer cells
in the liver or fixed macrophages in the spleen take up the
liposomes before they can reach their intended target. Capture by
the RES has rendered selected delivery of the liposomes to target
tissues or cells very difficult.
[0006] In addition to capture by the RES, the liposomes are subject
to electrostatic, hydrophobic, and Van der Waals interactions with
plasma proteins. These interactions result in destabilization of
the liposomes leading to rapid clearance of the vesicles from
circulation, often before reaching their target.
[0007] Also, in addition to cellular and protein interactions with
the liposomes, difficulties have arisen in producing liposome
encapsulating certain drugs because of the drugs' interactions with
the phospholipids of the liposomes. For example, anthracyclines
have exhibited a surfactant or detergent-like effect on the
phospholipid vesicle bilayer that causes leakage and creates
liposome vesicle instability. Thus, liposomes unstable to the
circulation environment and/or its content will leak the
antineoplastic agent prematurely before reaching the tumor site. As
a result of the "leaky" liposomes and the resulting devastating
toxicities, scientists have tried to develop long-circulating
liposomes that are able to extravasate to tumor sites, which are
highly vascular in nature.
[0008] Since most commonly used anti-cancer drugs are not
specifically toxic to tumor cells and are toxic to all tissues they
contact, they create undesirable side effects as a result of their
interactions with normal tissues. For example, Doxorubicin
hydrochloride is one of the most commonly used cytotoxic
anthracycline antibiotics used in cancer chemotherapy and has been
shown to have activity against a wide variety of neoplasms.
Doxorubicin hydrochloride is effective in the treatment of many
solid tumors and leukemias. It is particularly effective in the
treatment of breast cancers involving polytherapies. Doxorubicin
hydrochloride is protocol therapy for AIDS related Kaposi's
sarcoma. Doxorubicin hydrochloride also has notable activity
against tumors of the ovaries, lung, testes, prostate, cervix, head
and neck, oestrogenic sarcomas and Ewing's sarcoma.
[0009] Conventional compositions of Doxorubicin hydrochloride are
available as freeze-dried product or as a solution of Doxorubicin
hydrochloride in water. Freeze-dried product requires
reconstitution with Water for Injection before administration. Both
these marketed products have been associated with a number of
toxicities when administered intravenously. Severe myelosuppression
is usually the dose limiting factor. Other toxicities include
nausea and vomiting, alopecia, mucositis (including stomatitis and
esophagitis) and cardiotoxicity, which may limit Doxorubicin
hydrochloride use. Doxorubicin hydrochloride is a potent vesicant
that may cause extravasation and necrosis at the injection site or
at any site that the skin is exposed. "Doxorubicin flare" is not
uncommon and is characterized by erythematous streaking at the
injection site. "Doxorubicin flare" usually subsides in about a
half an hour.
[0010] The mechanism of action of Doxorubicin hydrochloride is not
known exactly but many possibilities have been studied and
described. The primary mechanism involves the ability of
Doxorubicin hydrochloride to intercalate DNA. The integrity of the
DNA is significantly compromised and commonly results in altered
DNA functions. Single and double strand brakes are also common due
to Doxorubicin hydrochloride intercalation with DNA. Another
mechanism of Doxorubicin hydrochloride involves its ability to
generate free radicals that induce DNA and cell membrane damage.
Doxorubicin hydrochloride also inhibits topoisomerase II, rendering
the reproduction of DNA ineffective.
[0011] Some of the resulting toxic affects of Doxorubicin
hydrochloride include cardiac toxicity, anaphylactic reaction,
emetogenicity, myelosuppression, muccocytis, skin toxicity,
alopecia, and toxicity to the injection sight. (Cancer
Investigation, 19 (4): 424-436 (2001)). In theory, prolonged
circulation systems (slow release) that effectively deliver and
release a drug to tumors and the near vicinity of tumor cells are
more advantageous. Thus, it is desirable to have a stable liposome
capable of encapsulating agents, such as Doxorubicin hydrochloride,
that do not prematurely release their contents to healthy or
non-cancerous tissues.
[0012] Several approaches taken in an effort to increase the
circulation time of liposomes and thus ensure delivery of the
liposome contents to the target tissue include the following:
masking the liposomes from the reticuloendothelial system
recognition using a sialic acid residue coating (U.S. Pat. No.
4,501,728); rigidifying the liposome membrane with sphingomyelin or
neutral phospholipid with predominantly saturated acyl chains
containing 5 to 20% glycolipid (U.S. Pat. No. 4,920,016); forming
liposomes with a 3-80 fold higher drug to lipid ratio than
traditional liposome preparations in a 3-compartment system of the
agent, bilayers, and release inhibiting buffer containing citric
acid (U.S. Pat. No. 6,083,530); incorporating cholesterol in the
liposome (Alberto A. Gabizon, Cancer Investigation, 19(4) 424-436
(2001)); and derivatizing the phospholipid with polyethylene glycol
(pegylated liposomes) (U.S. Pat. Nos. 5,013,556 and 6,132,763).
[0013] Unfortunately, the above approaches have shown only limited
potential to extend the circulation time of the liposomes in vivo.
For example, it has been determined that masking the liposome with
sialic acid only had limited ability to extend the circulation half
lives of in vivo liposomes. (U.S. Pat. No. 4,920,016). To overcome
these problems, scientists have coated the liposome surface with a
hydrophilic polymer such as polyethylene glycol (PEG) to prevent
adsorption of various blood plasma proteins to the liposome
surface. (See e.g. U.S. Pat. No. 5,013,556, and U.S. Pat. No.
5,676,971). These pegylated liposomes have been called sterically
stabilized liposomes or stealth liposomes. The pegylated liposomes
appeared to reduce some of the toxic effects caused by the release
of their contents, but, unfortunately, new toxic effects appeared
because of the presence of the polyethylene glycol. For example,
the liposomal preparations containing pegylated phospholipids have
lead to skin toxicity generally known as "Hand-Foot syndrome,"
which results in skin eruptions/ulcers on the palms of the hands
and soles of the feet. (Kenneth B. Gordon, Cancer, Vol. 75(8),
1995, 2169-2173).
[0014] Another disadvantage with pegylated liposomes is the
presence of large molecules (PEG) on the liposomal surface may
reduce the interactions of liposomes with cells and hinder entry of
liposomes into the tumor tissue, thereby possibly reducing the
accumulation of liposomal drug in the tumor tissue. (Clinical
Cancer Research, (5), 1999, 3645 -3652)
[0015] Thus, there remains a need for stable, long circulating
liposomes that do not cause such deleterious effects such as the
"Hand-Foot syndrome" as well as methods of manufacturing such
liposomes and compositions based on them. The present invention
meets this need, as well as provides for methods of treatment of
various conditions by administering the liposomes of the present
invention.
SUMMARY OF THE INVENTION
[0016] The present invention provides a process for the manufacture
of long circulating non-pegylated liposomes; the process comprising
dissolving one or more phospholipids, a sterol in a solvent or
mixture of solvents; removing the said solvents before or after
hydrating the lipids by addition of a aqueous hydration media to
form non-pegylated liposomes; wherein the amount of the aqueous
hydration media used is in the range of 10 to 35 ml for each mmole
of phospholipid present in the lipid solution.
[0017] Preferably the amount of aqueous hydration media used is 30
ml for each mmole of phospholipid in the lipid solution.
[0018] The present invention further provides a process for
manufacture of long circulating non-pegylated sized liposomes
comprising dissolving one or more phospholipids, and a sterol in a
solvent or mixture of solvents; removing said solvents before or
after hydrating the phospholipids by addition of an aqueous
hydration media to form non-pegylated liposomes; wherein the amount
of the aqueous hydration media used is in the range of 10 to 35 ml
for each mmole of phospholipid present in the lipid solution;
sizing the non-pegylated liposomes to about 0.06 .mu.m to 0.1 6
.mu.m to form a liposomal composition; and removing extra-liposomal
hydration salt from the liposomal composition using
sucrose-histidine buffer solution to form non-pegylated sized
liposomes.
[0019] The process of manufacture of the non-pegylated liposomes
may further comprise loading the liposomes with a therapeutic or
diagnostic agent. Preferably therapeutic agent is an antineoplastic
agent such as Doxorubicin hydrochloride, Daunorubicin
hydrochloride, and Epirubicin hydrochloride. Doxorubicin
hydrochloride is more preferred.
[0020] Preferably the molar ratio of phospholipid to sterol is from
about 1:0.1-1:2 and is more preferably about 1:0.7
[0021] A preferred aqueous hydration media comprises ammonium
sulfate and sucrose, and the concentration of ammonium sulfate in
the aqueous hydration media is not less than 125 mmoles/liter.
[0022] Preferred phospholipids have a phase transition temperature
of about 40.degree. C. to 60.degree. C., have a fatty acid chain of
a minimum of sixteen carbons and are selected from the group
consisting of Distearoyl phosphatidylcholine (DSPC), Dipalmitoyl
phosphatidylcholine (DPPC), Hydrogenated soya phosphatidylcholine
(HSPC) and derivatives of such phospholipids. A preferred
phospholipid is distearoyl phosphatidylcholine (DSPC) and a
preferred sterol is cholesterol.
[0023] The process may also involve sizing of the non-pegylated
liposomes. They are preferably sized by extrusion successively
through filters having a pore size of 0.4 .mu.m to 0.05 .mu.m.
[0024] Another embodiment of the present invention provides for
liposomes obtainable by the process described herein. Liposomes of
the present invention have the ingredients in the concentrations
and proportions described above in the process for the manufacture
thereof and the average size liposomes so obtained is 0.6 .mu.m to
0.16 .mu.m.
[0025] The present invention also provides for a long circulating
non-pegylated liposomal doxorubicin composition for parenteral
administration comprising, doxorubicin non-pegylated liposomes,
histidine hydrochloride, and sucrose; wherein the doxorubicin
non-pegylated liposomes comprise distearoylphosphatidyl choline,
cholesterol, sucrose in addition to doxorubicin hydrochloride;
wherein the liposomes have an average size 0.06 .mu.m to 0.160
.mu.m; and wherein the non-pegylated doxorubicin liposomes have a
circulation time in blood at least 25 times longer than that
obtained with ADRIAMYCIN when tested in Swiss albino mice at
equivalent doses.
[0026] Doxorubicin hydrochloride concentration encapsulated in the
liposomes is from is 1 to 10 mM, and preferably is from 3mM to 7mM,
more preferably 6.9mM and most preferably 3.45mM.
[0027] The molar ratio of distearoylphosphatidyl choline to
cholesterol is from 1:0.6 to 1:0.8; preferably 1:0.7.
[0028] The molar ratio of doxorubicin hydrochloride to
distearoylphosphatidyl choline is preferably from 1:2 to 1:15; and
more preferably 1:3.5.
[0029] The sucrose concentration is preferably from 0.1M to 0.5M,
and more preferably from 0.25M to 0.3M.
[0030] The concentration of histidine hydrochloride is from 1mM to
100mM, preferably from 8 to 12 mM, and more preferably about 10
mM.
[0031] The preferred average size of the liposomes is from 0.08
.mu.m to 0.12 .mu.m.
[0032] An exemplary composition is the doxorubicin hydrochloride
present at 2 mg/ml; and the molar ratio of doxorubicin to
phospholipid is about 1:3.5; and the ratio of phospholipid to
cholesterol is about 1:0.7.
[0033] Another exemplary composition comprises doxorubicin
hydrochloride present at 4 mg/ml and the molar ratio of doxorubicin
to phospholipid is about 1:3.5 and the ratio of phospholipid to
cholesterol is about 1:0.7. The circulation time (t1/2) of the
composition in blood is preferably more than 40 times longer than
that obtained with ADRIAMYCIN when tested in Swiss albino mice at
equivalent doses.
[0034] The present invention also provides a process for
manufacture of long-circulating non-pegylated liposomal doxorubicin
compositions.
[0035] The present invention also methods for reducing tumor growth
comprising administering the compositions of the present
invention.
DETAILED DESCRIPTION OF THE INVENTION
[0036] The present invention provides stable, long circulating,
non-pegylated liposomes, as well as a method of manufacture
thereof. Pegylated liposomes are liposomes coated with
polyethyleneglycol (PEG). The surface of the liposome is decorated
with several thousand strands of PEG, a process called
"pegylation." The PEG strands make the surface of the liposome
"hairy," and this prevents the rapid absorption of liposomes to the
surface of blood proteins. The rapid absorption accelerates the
rapid removal from blood of liposomes. In contrast, the pegylated
liposomes are protected and are removed from blood at a much slower
rate. Compared with liposomes made without PEG, pegylated liposomes
are more stable and are less extensively taken up by cells of the
reticuloendothelial system (RES), and have a reduced tendency to
leak any encapsulated agent or drug while in circulation. For
example, the pharmacokinetics of PEG-liposomes encapsulating
doxorubicin is characterized by a long circulating half-life, slow
plasma clearance, and a reduced volume of distribution compared
with non-pegylated liposomal doxorubicin or free doxorubicin. The
long circulation and ability of pegylated liposomes to extravasate
through tumor vasculature results in localization of doxorubicin in
tumor tissue with the increased possibility of increased tumor
response because of enhanced drug accumulation especially in highly
angiogenic tumors. Also, the increased stability of pegylated
liposomes over conventional liposomes results in a decrease in
availability of drug in the tissue of sensitive organs and thereby
a decrease in toxicity and other adverse effects such as nausea,
vomiting, and alopecia. A serious side effect known as "Hand-Foot
syndrome," however, where skin eruptions or ulcers have been
observed on the palms of the hands and soles of the feet, have been
reported to result from clinical uses of the pegylated liposomes.
(Kenneth B. Gordon, Cancer, Vol. 75(8), 1995, 2169-2173). Another
disadvantage with pegylated liposomes is the presence of large
molecules (PEG) on the liposomal surface may reduce the
interactions of liposomes with cells and hinder entry of liposomes
into the tumor tissue, thereby possibly reducing the accumulation
of liposomal drug in the tumor tissue.
[0037] The process of the present invention provides stable, long
circulating, low toxicity non-pegylated liposomes that exhibit the
stability of the pegylated liposomes with the long circulation
half-life and reduced toxicity described above. However, since the
liposomes of the present invention do not require the use of PEG to
achieve the above results, they do not cause "Hand-Foot
syndrome."
[0038] In the process of the present invention, hydration of lipids
may be carried out before evaporation of the solvent or may be
carried out after evaporation of the solvent that is used for
dissolving lipids. Solvents suitable to the invention are organic
solvents in which the phospholipid can be dissolved. One skilled in
the art would appreciate commonly used and suitable solvents in the
manufacture of liposomes. Exemplary suitable solvents include but
are not limited to chloroform, methylene chloride, ethanol,
methanol, acetone.
[0039] When the hydration of lipids is carried out after
evaporation of the solvent, solvents such as chloroform, methylene
chloride are preferred solvents.
[0040] When the hydration of lipids is carried out before
evaporation of the solvent, water miscible solvents such as
ethanol, methanol, acetone are preferred solvents
[0041] When hydration is carried out after evaporation of the
solvent, the process comprises; forming a lipid film by evaporating
a solvent from a lipid solution comprising one or more
phospholipids, a sterol, and a solvent or a mixture of
solvents.
[0042] Evaporation of a solvent can be accomplished by any
evaporative technique such as, but not limited to, evaporation by
passing a stream of inert gas over the solution, by heating, by
vacuum, or by heating under vacuum. Commonly, rotary evaporator
flasks are employed.
[0043] When the hydration is carried out before the evaporation of
solvent, the process comprises evaporation of the solvent from the
aqueous liposomal suspension containing solvent. Evaporation of a
solvent can be accomplished by any evaporative technique such as,
but not limited to, evaporation by passing a stream of inert gas
over the solution, by heating, by vacuum, or by heating under
vacuum. Commonly, rotary evaporator flasks are employed. After a
solvent or mixture of solvents is evaporated, only the liposomes
remain in the aqueous suspension form.
[0044] Any phospholipid suitable to prepare liposomes may be used
in the present invention. Suitable phospholipids include those that
tend to decrease permeability of the liposomal membrane. Liposomes
containing phospholipids with long fatty acid chains are more
suitable and result in a slower release of agent than liposomes
comprised of phospholipids having shorter fatty acid chains. As the
carbon chain length of the fatty acid increases, the phase
transition temperature also increases. Liposomes comprised of
phospholipids with higher phase transition temperature release
their contents slower than liposomes comprised of lower phase
transition phospholipids. Higher phase transition temperatures
enable slow releasing of the contents from inside the liposomes
into the blood stream as the phospholipid membranes are
semipermeable. Other phospholipid characteristics that effect
membrane permeability and stability include degree of saturation
and charge.
[0045] Preferably, liposomes of the present invention contain
neutral lipids. It is preferred that the neutral lipids have a
phase transition temperature of 40.degree. C. to 65.degree. C. and
more preferably of about 50.degree. C. to 54.degree. C. Preferable
phospholipids have a fatty acid chain of at least sixteen
carbons.
[0046] Suitable phospholipids of the present invention include, but
are not limited to, Distearoyl phosphatidylcholine (DSPC), or
Dipalmitoyl phosphatidylcholine (DPPC), Hydrogenated soya
phosphatidylcholine (HSPC) or derivatives of such phospholipids.
Phosphatidylcholines are preferred neutral lipids. A preferred
phospholipid is 1,2,-Distearoly-sn-glycerol-3- -phosphocholine,
which is commonly known as distearoyl phosphatidylcholine (DSPC).
The molecular weight of DSPC is 790 and has the molecular formula
of C.sub.44H.sub.88NO.sub.8P.
[0047] Sterols are incorporated into liposomes along with
phospholipids to alter rigidity and permeability of liposome
membranes. An exemplary sterol is cholesterol and derivatives or
analogs thereof. Cholesterol tends to increase rigidity and
decrease permeability of liposomal membranes. Cholesterol is an
amphipathic molecule and inserts itself into the phospholipid
membrane with its hydroxyl groups orientated towards the aqueous
surface. Cholesterol is incorporated in a concentration that
provides optimum permeability to the liposome membrane, but also
maintains the rigidity of the membrane. The selection of
phospholipid to cholesterol ratio defines the rate of dissolution
of the contents from the liposomes. Liposomes of the present
invention have a molar ratio of phospholipids to sterol ranging
from 1:0.1 to 1:2. Preferably the range is from 1:0.5 to 1:1.5. A
preferable molar ratio of phospholipids to sterol when distearoly
phosphotidyl choline (DSPC) is the phospholipid and cholesterol is
the sterol is from 1:0.6 to 1:0.8. A preferred molar ratio is about
1:0.7.
[0048] The solvent or mixtures of solvents are evaporated under
vacuum. In the process when the hydration is carried out after
removing the solvents, the lipid film formed is hydrated with an
aqueous hydration media to form liposomes. The aqueous hydration
media is added to the film with agitation or under mixing to
hydrate the lipid film and form liposomes. One skilled in the art
would appreciate suitable aqueous hydration medias to employ.
Preferable aqueous hydration medias contain buffers/salts so as to
be available to establish a chemical gradient later in the process
to assist in loading various agents into the liposomes. Exemplary
hydration medias include, but are not limited to, ammonium
hydroxide, ammonium sulfate, ammonium carbonate, and ammonium
bicarbonate. A preferred aqueous hydration media contains ammonium
sulfate. Also, the aqueous hydration media contain an iso-osmotic
agent, such as but not limited to sucrose, sodium chloride,
dextrose, or mannitol. It is preferable that the iso-osmotic agent
is non-reactive with other contents of the solution and the
liposomes themselves. The iso-osmotic agent is preferably sucrose
since it is least reactive. When the aqueous hydration media
contains ammonium sulfate, preferably the iso-osmotic agent is
sucrose. Sucrose helps in protecting and rigidifying the liposomal
membrane and also to maintain the isotonicity of the liposomal
composition.
[0049] The volume of the aqueous hydration media is
controlled/reduced as compared to amounts of hydration media used
in conventional liposome and pegylated liposome manufacture. By
reducing the volume of aqueous hydration media, the phospholipids
can pack tighter together resulting in a thicker liposome membrane
or "shell." The thicker "shell" provides for stable,
long-circulating, slow release and decreased toxicity of the
liposome contents without the need for PEG. The smaller the volume
of hydration media used, the tighter the phospholipids will pack
together and the thicker the shell will become. By
"controlled/reduced" it is meant that the volume of aqueous
hydration media used in the present invention is less than
previously known or accepted amounts of aqueous hydration media.
Using a preferred reduced volume of hydration media (i.e. 30 ml for
each mmole of phospholipid) and a preferred concentration of
cholesterol, the resulting liposomal composition would have a rigid
phospholipid bilayer.
[0050] This reduction in hydration volume can also be viewed in
terms of the ratio of volume of buffer used per moles of
phospholipid present in the lipid solution. In the present
invention, the amount of aqueous hydration media used is in the
range of 10 to 35 ml for each mmole of phospholipid present in the
lipid solution. Preferably the volume of aqueous hydration media is
between 20-30 ml for each mmole of phospholipid present in the
lipid solution. More preferably, the volume of aqueous hydration
media is 30 ml for each mmole of phospholipid used in the lipid
solution.
[0051] Liposomes are sized appropriately. One skilled in the art
would appreciate known methods of liposome sizing. Homogenization
under pressure is one such method. Another suitable method includes
extruding the liposomes through filters with a pore size to match
the desired liposome size. Because the liposomes of the present
invention have a tighter packed membrane, sizing tends to be more
difficult than with conventional liposomes. Thus, they are
preferably sized through a series of filters with increasingly
smaller pore size. For example, following hydration, liposomes are
initially passed through a filter having a pore size of 0.40 .mu.m
followed by successively smaller pore sized filters of about 0.05
.mu.m. The resulting liposomes have an average size range from 0.06
.mu.m-0.2 .mu.m. A preferred average size range is from 0.08 .mu.m
to 0.12 .mu.m.
[0052] Extraliposomal salt in the hydration media is removed or
washed from the liposomes. Dialysis using a dialysis medium is an
exemplary method of removing extraliposomal hydration media salt.
Any suitable buffer solution may be used in the dialysis. Removal
of extraliposomal salt present in the liposomal composition creates
an inside-to-outside chemical gradient across the liposomal
membrane, which is later called upon for loading of the liposomes.
Other suitable means to remove the extraliposomal salt includes
ultrafiltration or column chromatography.
[0053] The liposomes of the present invention provide a long
circulating, slow release delivery mechanism for therapeutic or
diagnostic agents. Any known method can be used to load the
liposomes with a desired therapeutic or diagnostic agent. Exemplary
methods include adding the agent to the lipid film before hydration
of the lipid film, incorporating the agent directly into the
hydration media, by pH gradient, or by chemical gradient. A
preferred method involves loading an agent using a chemical
gradient. When the liposomes are loaded by active loading process,
the drug solution is admixed with the blank liposomal suspension at
a temperature higher than or equivalent to phase transition
temperature of the phospholipids.
[0054] Using a chemical gradient, the amount of agent can be
readily controlled and once the agent is loaded inside the
liposomes, the leakage into the extraliposomal media is minimal. In
addition, if a hydration media containing a buffer/salts is used in
the hydration step, the creation of such a gradient becomes very
feasible after removing the extraliposomal hydration media salt as
described above. One such exemplary hydration media that may be
used to create a chemical gradient useful in liposome loading
contains ammonium sulfate. However, hydration with Ammonium sulfate
solution rendered isotonic with sodium chloride (See U.S. Pat. No.
5,316,771) results in liposomes which leak on storage. The free
drug content of the liposomal composition increases on storage,
which in turn increases the toxicity. Hence there is a need to
strengthen the liposomal membrane. The present invention thus
provides the concomitant use of an iso-osmotic agent that is
non-reactive with other ingredients of the solution and the
liposomes themselves in the hydration media. Preferably the
iso-osmotic agent is sucrose. It was found that use of sucrose is
protective for liposomal membranes. Sucrose helps in protecting and
rigidifying the liposomal membrane and also to maintain the
isotonicity of the liposomal composition. Liposomal membranes have
been protected for dehydration before freeze drying by use of
saccharides such as trehalose, sucrose, maltose (U.S. Pat. No.
4,880,635).
[0055] The present invention thus provides using sucrose with
ammonium sulfate as a hydration medium giving liposomes that are
more rigid and that do not leak the agent encapsulated in them on
storage. With the addition of sucrose to the hydration medium,
sucrose remains inside and outside surface of the liposomal
membrane hardening both sides of the liposomal membrane, thereby
reducing the leakage of the drug. It is preferable that the
concentration of sucrose in the hydration media is from 0.1M to 0.5
M. A concentration of 0.25M to 0.3M is preferred.
[0056] The concentration of ammonium sulfate in the hydration media
plays a vital role on drug leakage from the liposomes. Ammonium
sulfate solution in a concentration less than 125mM whenever used
for hydration for forming liposomes showed the drug leakage on
storage. Thus in a preferred method of manufacture, the
concentration of ammonium sulfate in hydration media is greater
than 125mM, which in turn produces liposomal compositions with
reduced leakage on storage. Thus, in a preferred method the
concentration of ammonium sulfate solution is not less than 125
mmole/liter, and the hydration media contains sucrose.
[0057] When dialysis is performed, it removes the extraliposomal
salt, i.e. ammonium sulfate, but does not remove intra-liposomal
ammonium sulfate, thus causing the inside-to-outside chemical
gradient across the liposome membrane.
[0058] There are many suitable buffer solutions that can be used
both to load the drug into the liposomes and to dilute the
resulting liposomal composition to a desired concentration of the
drug. Since liposomes primarily contain phospholipids, which are
stable at around neutral pH of about 6.0 to 8.0, buffer solutions
used to load and dilute liposomes should also have a neutral pH.
Also, ideally the buffer solution should be suitable for parenteral
preparations. Some of the most common buffer solutions used in
parenteral preparations, which are suitable in the present
invention for loading the drug into the liposomes and for dilution
of the liposomal composition, are glycine, phosphate, citrate,
acetate, and histidine buffers. Histidine buffer solution is
preferable as it has the most stable pH in the neutral range.
Preferably, the buffer solution comprises sucrose and histidine
hydrochloride in a molar ratio from 29:0.1 to 29:10, more
preferably about 29:1. Use of sucrose helps in protecting and
rigidifying the liposomal membrane and also in maintaining the
isotonicity of the liposomal composition.
[0059] After the liposomes are loaded, any untrapped agent is
removed. Suitable methods include, but are not limited to, gel
filtration chromatography, dialysis, treatment with microporus
styrene/divinylbenzene copolymer resin (DOWEX) and subsequent
filtration. DOWEX treatment is a preferred method because of its
ease of use. When dialysis is used, it is preferably performed in
the same manner as described above when removing extraliposomal
hydration media salts.
[0060] As discussed above, by controlling or reducing the amount of
aqueous hydration media, the resulting liposomes have an increased
phospholipid content per unit volume. Increase in phospholipid
content increases liposome stability, decreases permeability, and
thus slows the release of any entrapped agent.
[0061] Suitable agents for loading into liposomes of the present
invention are water soluble amphipathic compounds with ionizable
groups. Amphipathic agents exhibit both hydrophilic and lipophilic
characteristics and may be a therapeutic or diagnostic agent.
Therapeutic agents may be any desired agent and include
antineoplastic agents.
[0062] An antineoplastic agent is a drug that prevents, kills, or
blocks the growth and spread of cancer cells. There are many
suitable antineoplastic agents some of which include Altretamine;
Asparaginase; BCG; Bleomycin sulfate; Busulfan; Carboplatin;
Carmustine; Chlorambucil; Cisplatin-cis-platimum,
cis-diammine-dichloroplatinum; Cladribine, 2-chlorodeoxyadenosine;
Cyclophosphamide; Cytarabine-cytosine arabinoside; Dacarbazine
imidazole carboxamide; Dactinomycin; Daunorubicin-daunomycin,
Daunorubicin hydrochloride; Dexamethasone; Doxorubicin, Doxorubicin
hydrochloride; Epirubicin; Etoposide-epipodophyllotoxin;
Floxuridine; Fluorouracil; Fluoxymesterone; Flutamide; Fludarabine;
Goserelin; Hydroxyurea; Idarubicin HCL; Ifosfamide-Isophosphamide;
Interferon alfa; Interferon alfa 2a; Interferon alfa 2b; Interferon
alfa n3; Irinotecan; Leucovorin calcium; Leuprolide; Levamisole;
Lomustine; megestrol; Melphalan-L-phenylalanine mustard,
L-sarcolysin; Melphalan hydrochloride; Mechlorethamine, nitrogen
mustard; Methylprednisolone, Methotrexate-Amethopterin,
Mitomycin-Mitomycin-C; Mitoxantrone; Mercaptopurine, Paclitaxel;
Plicamycin-Mithramycin; Prednisone; Procarbazine;
Streptozocin-Streptozot- ocin; Tamoxifen; 6-thioguanine;
Thiotepa-triethylene thiophosphoramide; Vinblastine; Vincristine;
or vinorelbine tartrate. Preferred antineoplastic agent of this
invention include Doxorubicin hydrochloride, Daunorubicin
hydrochloride, and Epirubicin hydrochloride.
[0063] The present invention also provides for loading the
liposomes with diagnostic agents including, but not limited to, MRI
(magnetic resonance imaging) contrast agents (also called
paramagnetic agents) used to help provide a clear picture during
MRI. MRI is a special kind of diagnostic procedure that uses
magnets and computers to create images or "pictures" of certain
areas inside the body. Unlike x-rays, it does not involve ionizing
radiation. Exemplary MRI diagnostic agents include Gadodiamide;
Gadopentetate; Gadoteridol; Gadoversetamide,
Gd:diethylenetriaminepentace- dic acid chelate (Gd-DTPA) (U.S. Pat.
No. 6,132,763).
[0064] Once liposomes are loaded, and the unencapsulated,
therapeutic/diagnostic agent is removed, the liposomal composition
may be aseptically filtered for sterilization making it suitable
for parenteral administration. Ideally the filter is at least a 0.2
.mu.m filter. The liposomal composition is then filtered into a
sterile depyrogenated bulk container. Subsequently the sterile
composition is filled aseptically into sterile depyrogenated
smaller containers such as glass vials. The air in the headspace of
the container is removed by purging with an inert gas, such as
nitrogen and the containers are sealed. By "suitable for parenteral
administration" it is meant that the composition is sterile,
isotonic and controlled for bacterial endotoxins.
[0065] The present invention also provides for stable,
long-circulating, low toxicity non-pegylated liposomes. The
liposomes are preferably manufactured by the methods described
herein. The liposomes of this invention are long circulating
non-pegylated liposomes that have a blood circulation half-life of
at least 25 times longer than conventional non-liposomal
formulations (ADRIAMYCIN), when tested in Swiss albino mice at
equivalent doses. A preferred blood circulation half-life is about
40 times longer than that obtained with ADRIAMYCIN.
[0066] Non-pegylated liposomes of the present invention are
comprised of a phospholipid and cholesterol. Acceptable ratios of
phospholipid to cholesterol are described above and are preferably
at a molar ratio of about 1:0.1 to 1:2. A preferred molar ratio of
phospholipid to sterol is about 1:0.7. Phosphatidyl cholines are
preferred phospholipids and disteraroyl phosphatidylcholine (DSPC)
is especially preferred.
[0067] The non-pegylated liposomes may be loaded with a diagnostic
or therapeutic agent. Such agents are known and discussed above.
Non-pegylated liposomes of the present invention are preferably
loaded using a chemical gradient as discussed above.
[0068] A preferred non-pegylated liposome of the present invention
is loaded with doxorubicin hydrochloride and is prepared using
methods described above. In one embodiment, when loading
doxorubicin hydrochloride using the active loading procedure
described above, the drug is dissolved in a suitable buffer
solution (as described above) before loading to get a concentration
of at least 25mM. When the active loading process involves an
ammonium sulfate gradient, the ammonium sulfate reacts with
doxorubicin hydrochloride to form doxorubicin sulfate. Doxorubicin
sulfate is insoluble and remains inside the liposomes after
loading. Once any unentrapped or free drug is removed from loaded
liposomes, the drug loaded liposomes are diluted using aqueous
buffer solution to achieve the required drug concentration. The
preferred buffer solution used is sucrose-histidine buffer solution
as discussed previously.
[0069] An exemplary non-pegylated liposomal doxorubicin composition
contains 2 mg/ml doxorubicin hydrochloride. Another exemplary
non-pegylated liposomal doxorubicin composition contains 4 mg/ml
doxorubicin hydrochloride. Using methods of the present invention,
the doxorubicin may be loaded into non-pegylated liposomes at a
concentration twice of that desired in the final desired
composition. Then the loaded liposomes may be diluted with a
suitable buffer solution (as described above) to achieve the
desired concentration of doxorubicin per ml of liposomal
composition. On dilution, the external media in which the liposomes
are suspended is diluted, whereas the drug inside the liposomes
remains undiluted.
[0070] In a preferred embodiment, the molar ratio of doxorubicin
hydrochloride to phospholipids is from about 1:2 to about 1:15. A
preferred molar ratio is about 1:3.5.
[0071] The present invention also provides non-pegylated liposomal
doxorubicin compositions. The composition comprises non-pegylated
liposomes as described above in suitable pharmaceutically
acceptable carriers, which are known in the art. The liposomes have
been loaded with doxorubicin hydrochloride. The compositions are
suitable for parenteral administration, and are long
circulating.
[0072] One embodiment provides a long circulating non-pegylated
liposomal doxorubicin compositions for parenteral administration.
The liposomal composition comprises non-pegylated doxorubicin
liposomes in a pharmaceutically acceptable carrier. Suitable
pharmaceutically acceptable carriers are known in the art. In a
preferred pharmaceutical composition, the concentration of
Doxorubicin hydrochloride varies from 1mM to 10mM, more preferably
about 6.9mM, and the most preferable is about 3.45mM. The Molar
concentration of phospholipids varies from 10mM to 15mM of the
parenteral composition. A more preferred content is about
12.15mM.
[0073] The composition further comprises distearoylphosphatidyl
choline, cholesterol, histidine hydrochloride, and sucrose.
Preferably the liposomes have an average size from 0.06 .mu.m-0.16
.mu.m.
[0074] Preferably the doxorubicin hydrochloride content is 1-10mM
and more preferably the doxorubicin hydrochloride content is
3.45mM.
[0075] In the compositions of the present invention, the molar
ratio of distearoylphosphatidyl choline to cholesterol is from
1:0.6-1:0.8, and is preferably 1:0.7.
[0076] In the compositions of the present invention, the molar
ratio of doxorubicin hydrochloride to distearoylphosphatidyl
choline is from 1:2-1:10, preferably from 1:2-1:8 and more
preferably 1:3.5.
[0077] The sucrose content is from 0.1M-0.5M, and more preferably
is 0.25M to 0.3M.
[0078] In the compositions of the present invention the content of
histidine hydrochloride is from 1mM to 100mM, preferably 8 -12mM,
and more preferably 10mM.
[0079] In the compositions of the present invention, the liposomes
have an average size of 0.08 .mu.m-0.12 .mu.m.
[0080] In one embodiment of the present invention, the doxorubicin
hydrochloride is present at 4 mg/ml, and the molar ratio of
doxorubicin to DSPC is 1:3.5, and the ratio of DSPC to cholesterol
is 1:0.7.
[0081] In yet another embodiment of the present invention, the
doxorubicin hydrochloride is present at 2 mg/ml, and the molar
ratio of doxorubicin to DSPC is 1:3.5, and the ratio of DSPC to
cholesterol is 1:0.7.
[0082] The doxorubicin liposomes in the compositions preferably
have a half circulation time (t1/2) in blood at least 40 times
longer than ADRIAMYCIN when tested in Swiss albino mice at
equivalent doses.
[0083] Another embodiment of the present invention provides a
method for reducing tumor growth by administering non-pegylated
liposomal doxorubicin composition. This method involves
administering a therapeutically effective amount of a non-pegylated
liposomal doxorubicin composition of the present invention. As
non-pegylated liposomal doxorubicin composition have a prolonged
circulation time, exhibit decreased toxicity and do not present
"Hand-Foot Syndrome" issues, they provide a viable treatment for
reducing tumor growth. A skilled practitioner would be able to use
the data presented herein as well as common knowledge of dosage
amounts, dosage times, and routes of administration, to treat an
individual having a tumor susceptible to treatment by doxorubicin
hydrochloride with the non-pegylated doxorubicin liposomes of the
present invention. The compositions of the present invention having
2 mg/ml and 4 mg/ml doxorubicin hydrochloride strengths are useful
for treatment of reducing tumor growth.
[0084] The present invention also provides for a process for making
these compositions with the ingredients in the same proportions as
in the compositions. The process comprises: a process for
manufacture of a long circulating non-pegylated liposomal
doxorubicin composition for parenteral administration
comprising
[0085] (a) dissolving lipids comprising
Distearoylphosphatidylcholine (DSPC) and cholesterol in a single
solvent or in a mixture of solvents,
[0086] (b) removing said solvents before or after hydrating the
lipids by addition of an aqueous hydration media to form liposomes
in a liposomal composition, wherein said aqueous hydration media
comprises ammonium sulfate and sucrose, and wherein the aqueous
hydration media is added in quantities in the range of lOml to 35
ml per each mmole of DSPC;
[0087] (c) sizing the liposomes in the liposomal composition
obtained at the end of step (b), to about 0.060 .mu.m-0.16
.mu.m;
[0088] (d) removing extraliposomal ammonium sulfate from the
liposomal composition that has undergone sizing at step (c), using
a sucrose-histidine buffer solution comprising histidine
hydrochloride and sucrose;
[0089] (e) dissolving doxorubicin hydrochloride in said
sucrose-histidine buffer solution to obtain a solution of at least
25mM doxorubicin hydrochloride concentration;
[0090] (f) admixing doxorubicin hydrochloride solution obtained at
step (e) and the liposomal composition obtained at the end of step
(d) to obtain doxorubicin hydrochloride loaded liposomal
composition;
[0091] (g) removing extraliposomal doxorubicin hydrochloride from
the liposomal composition by a process selected from the group
consisting of tangential flow filtration, column chromatography and
treatment with resins;
[0092] (h) making up the volume of the liposomal composition
obtained at the end of step (g) with said sucrose-histidine buffer
solution to obtain a liposomal composition of a desired
concentration of doxorubicin hydrochloride;
[0093] (i) filtering aseptically, the liposomal composition through
a sterile 0.2.mu. sterilising grade filter into a sterile container
to obtain said liposomal doxorubicin composition.
[0094] The concentration of ammonium sulfate in the aqueous
hydration media is not less than 125mM.
[0095] Non-pegylated liposomes containing doxorubicin hydrochloride
of the present invention have shown decreased toxic effects as
compared to conventional doxorubicin hydrochloride formulations
(ADRIAMYCIN) and pegylated liposomal doxorubicin hydrochloride
formulations (CAELYX). Table 1, below, provides the results of
acute toxicity and pharmacokinetic studies in mice. Non-pegylated
doxorubicin liposomes of the present invention as manufactured by
the parameters set forth in Example II were compared to
commercially available pegylated liposomal doxorubicin formulation,
CAELYX and ADRIAMYCIN. The LD.sub.50 for the non-pegylated
doxorubicin liposomes of the present invention is higher than
ADRIAMYCIN and CAELYX, thus demonstrating that the non-pegylated
doxorubicin liposomes of the present invention have lower
toxicity.
1TABLE 1 ACUTE TOXICITY AND PHARMACOKINETIC STUDIES IN MICE
Parameters Example II CAELYX ADRIAMYCIN LD.sub.50 (mg/kg) 16.13
13.5 10.29 MTD (mg/kg) 8 8 5 C.sub.max (.mu.g/ml) 267.54 285.74
26.8 T.sub.max (hours) 0.085 0.085 0.085 Kel 0.0997 0.07109
4.851811 T.sub.1/2 (hours) 6.948 9.748 0.143 AUC (.mu.g-h/ml)
1694.024 2083.215 1.244 Vd (ml) 1.480 1.688 41.42 Vd (ml/kg) 59.20
67.52 1656.79 Cl (ml/h) 0.15 0.12 200.96 Abbreviations: MTD =
maximum tolerated dose; C.sub.max = maximum concentration of drug
achieved in the plasma; T.sub.max = time taken to achieve the
maximum concentration of drug in the plasma; Kel = elimination
constant; T.sub.1/2 = time required for the drug concentration in
the plasma to get decreased by 50%; AUC = area under
"concentration" vs. "time"curve; Vd = volume of distribution; Cl =
clearance rate of drug
[0096] Non-pegylated doxorubicin liposomes of the present invention
were used on MCF-7 human breast tumor implanted in mice. The
results are provided in Table 2, below. The difference in tumor
weight and effectiveness is measured by T/C % (test to control
percentage). In this study (Example VI), the highest ratio of T/C
using CAELYX is -78 at 12 mg/kg and -34.7 at 6 mg/kg, whereas using
the non-pegylated doxorubicin liposomes of the present invention,
the highest is -93.4 at 12 mg/kg and -89.43 at 6 mg/kg. These
results demonstrate that the non-pegylated doxorubicin liposomal
compositions of the present invention appear to be more effective
in reducing tumor weight than the currently marketed pegylated
liposomal formulation, CAELYX.
2TABLE 2 EFFECT ON MCF-7 HUMAN BREAST TUMOR IMPLANTED IN NUDE MICE
Average Tumor Weight (mg) Composi- tion of Example Composition
CAELYX Group Saline II of Example II CAELYX (12 mg/ Day Control (6
mg/kg) (12 mg/kg) (6 mg/kg) kg) 1 36.5 31.5 68.4 38.3 57.88 5 36.75
45.33 81.6 44.3 50.75 9 63.13 40.17 43.6 41.5 31.38 12 52.38 42.83
46.1 60.17 32 16 78.13 5.33 16.2 25 27.8 19 94 3.33 8 25 22.8 23
95.38 3.33 4.5 16 16.6 26 94.38 3.33 4.5 25 12.6 Wt. 43.4 -28.17
-63.9 -13.3 -45.2 T/C % NA -89.43 -93.4 -34.7 -78
[0097] Anti-tumor activity of non-pegylated doxorubicin liposomes
of the present invention against L1210 mouse leukemia cells was
tested. The results are provided in Table 3, below. The results of
this test (Example VI) show that non-pegylated doxorubicin
liposomal compositions of the present invention are as effective as
the pegylated liposomes (CAELYX).
3TABLE 3 ANTI-TUMOR ACTIVITY AGAINST L1210 MOUSE LEUKEMIA MODEL
Dosage Survival Mean Survival Group (mg/kg) Mice Time (Days) Time
(Days) T/C % Saline Control NA 1/5 17 16 NA 2/5 16 3/5 17 4/5 16
5/5 16 Example II 6 1/5 20 20.4 128 2/5 20 3/5 22 4/5 20 5/5 20
Example II 12 1/5 23 21.2 132 2/5 20 3/5 20 4/5 20 5/5 23 Caelzx
.RTM. 6 1/5 18 20.4 128 2/5 22 3/5 20 4/5 20 5/5 22 CAELYX 12 1/5
18 20.6 129 2/5 22 3/5 20 4/5 23 5/5 20 T/C %: Test to control
percentage
[0098] The above results in Tables 1-3 demonstrate that the
non-pegylated liposomal doxorubicin composition of the present
invention has a lower toxicity profile and a longer circulation
time and has proven efficacy of anti-tumor activity in-vivo against
MCF-7 and L1210 tumor models.
[0099] In order that those skilled in the art can more fully
understand this invention, the following examples, which describe
the preparation, characterization, and in vivo chemotherapeutic
application in an animal model of liposome formulations of this
invention, are set forth. These examples are presented solely for
purposes of illustration and are not intended to limit the present
invention in any way.
EXAMPLES
[0100] Doxorubicin hydrochloride used in these Examples was of
parenteral grade complying with US Pharmacopoeial specifications.
Phospholipids used in these Examples were of parenteral grade.
Cholesterol used in these Examples was complying with US
Pharmacopoeial specifications. Water used in these Examples was of
parenteral grade complying with Water for Injection specifications.
All other additives used in these Examples were of parenteral
grade. The entire processing was carried out in an area with a
controlled environment.
[0101] CAELYX (Pegylated liposomal Doxorubicin formulation)
manufactured by Ben Venue Laboratories, USA and ADRIAMYCIN
(Conventional non-liposomal Doxorubicin formulation) manufactured
by Pharmacia & Upjohn, USA were used in animal studies for
comparative evaluation with Non-pegylated liposomal Doxorubicin
formulations of the present invention. ADRIAMYCIN, which is also
referred to herein as "Conventional non-liposomal doxorubicin
composition" is a freeze dried sterile powder for injection, each
vial containing Doxorubicin hydrochloride 10 mg, Lactose 50 mg,
Methylhydroxybenzoate 1 mg. Before use, the freeze dried powder is
reconstituted with 5 ml of Water for Injection provided with the
pack.
[0102] For hematological testing, Cell Counter (Sysmex Automated
Hematology Analyzer-KX-21 was used.
Example I
Process of Making A Liposomal Composition Containing Doxorubicin
Hydrochloride
[0103] Lipid film formation: DSPC (1.565 g) and cholesterol (0.521
g) were dissolved one after the other in chloroform (40 ml) in a
rotary evaporator flask. They were mixed until a clear solution was
formed. The flask was connected to a Rotary evaporator and the
water bath temperature was adjusted to 60.degree. C. The solvent
was evaporated under vacuum to form thin film of lipids on the wall
of the flask. After releasing the vacuum, the flask was rotated for
approximately 5 minutes while passing nitrogen into the flask to
dry off any residual solvent.
[0104] Hydration: The lipid film in the flask was then hydrated
with 60 ml of aqueous hydration media containing ammonium sulfate.
The hydration media consists of 10.0 gm of Sucrose, 2.04 gm of
Ammonium sulfate, and 100 ml of water. The flask containing the
lipid film and hydration media was rotated for 30 minutes on a
water bath maintained at 65-68.degree. C. to form liposomes.
[0105] Size reduction of liposomes by extrusion: The liposomal
suspension obtained from above was sized by extruding successively
through filters having pore size from 0.4 .mu.m and to 0.05
.mu.m.
[0106] Development of ammonium sulfate gradient: The suspension of
the sized liposomes was dialyzed against a sucrose-histidine buffer
solution to remove extra-liposomal ammonium sulfate thereby
creating a chemical gradient. A tangential flow filtration system
fitted with a 300 KD cassette was used for the dialysis. The
absence of ammonium sulfate was tested using Nesseler agent.
[0107] The sucrose-histidine buffer solution used in the dialysis
and drug loading (below) is as follows: 170.0 gm of sucrose, 3.40
gm of histidine HCl, 1.7 Liters of water, and sodium hydroxide at a
quantity sufficient to adjust pH to 6.0 to 6.5.
[0108] Drug loading: In a round bottom flask, a 15 mg/ml solution
of Doxorubicin HCl in sucrose-histidine buffer solution (described
above) was prepared to load the liposomal preparation and to get
drug loaded liposomes having a concentration of 4 mg/ml of
doxorubicin hydrochloride. The sized and dialyzed liposomes from
above were added slowly to the round bottom flask and mixed for one
hour at 65.degree. C. The drug loaded liposomes were mixed with
DOWEX for 30 minutes to remove the unentrapped drug. The drug
loaded liposomes were diluted to a 2 mg/ml concentration using
sucrose-histidine buffer solution and then aseptically filtered
using a sterile 0.22 .mu.m membrane filter. The filtered liposomal
doxorubicin composition was then filled aseptically into sterile
depyrogenated glass vials and sealed under cover of nitrogen using
TEFLON coated rubber bungs.
Example II
LD.sub.50 Comparison of Pegylated Liposomal Doxorubicin
Formulations, Non-Liposomal Doxorubicin Composition, And
Non-Pegylated Liposomal Doxorubicin Composition of the Present
Invention
[0109] The following liposomal doxorubicin composition was
prepared. Each ml of the composition having:
4 DSPC 9.55 mg Cholesterol 3.15 mg Doxorubicin Hydrochloride 2.01
mg Sucrose 95 mg Histidine Hydrochloride 2 mg
[0110] The composition was prepared by the same procedure as in
Example I. Doxorubicin hydrochloride (216 mg) was dissolved in 14
ml of sucrose-histidine buffer solution and added to 40 ml of sized
liposomes and mixed for 1 hour. The resultant drug loaded liposomal
dispersion was then passed through a DOWEX column to remove
unentrapped drug.
[0111] The product obtained after passing through the DOWEX column
had the following characteristics:
[0112] Product Analysis
5 Total Doxorubicin HCl content 3.98 mg/ml Entrapped Doxorubicin
HCl content 3.94 mg/ml
[0113] The above product after dilution with histidine buffer to a
concentration of 2 mg/ml was analyzed for the following
parameters:
6 Appearance Red colored translucent liquid pH 6.1 Particle size
Average particle size 0.093 .mu.m DSPC content 9.55 mg/ml
Cholesterol content 3.15 mg/ml Doxorubicin HCl 2.01 mg/ml content
Bacterial endotoxins Less than 2.2 EU/mg of doxorubicin
hydrochloride. Sterility Sterile Sucrose content 9.35% Histidine
HCL content positive
[0114] This composition was subjected to acute toxicity studies in
mice. A LD.sub.50 comparison of "pegylated liposomal doxorubicin
composition" (CAELYX), "conventional non-liposomal doxorubicin
composition" (ADRIAMYCIN), and "non-pegylated liposomal doxorubicin
composition of the present invention" was performed.
7 Animals used Swiss albino mice of either sex. Weight range of
animal 20-22 gm. Number of groups 3 Number of animals per group
10
[0115] Animals were divided into 3 groups and each group comprised
of ten animals. GROUP 1 received Composition of Example II, GROUP 2
received CAELYX, and GROUP 3 received ADRIAMYCIN.
[0116] All animals received injections via the intravenous route.
The drug solutions were suitably diluted with dextrose (5% w/v)
solution before administering to the animals. The animals were then
observed for a period of 14 days. They were observed for any
clinical toxicity and mortality.
[0117] The LD.sub.50 values of the different Doxorubicin
compositions studied are provided in Table 1. The LD.sub.50 dose
was found to be 16.13 mg/kg whereas the LD.sub.50 dose for the
marketed conventional preparation (ADRIAMYCIN) was 10.29 mg/kg. The
LD.sub.50 for the marketed pegylated liposomal preparation CAELYX
was 13.5 mg/kg. These results show that non-pegylated liposomes of
the present invention have a reduced toxicity as compared to other
Doxorubicin formulations and to pegylated-liposomal Doxorubicin
formulations.
Example III
Comparison of Subacute Toxicity of "Non-Pegylated Liposomal
Doxorubicin Composition of the Present Invention With "Pegylated
Liposomal Doxorubicin Composition" (CAELYX) And "Conventional
Non-Liposomal Doxorubicin Composition" (ADRIAMYCIN)."
[0118]
8 Animals used Swiss albino mice of either sex Number of groups 11
Number of animals per group 8 Weight range of animal 19-23 gms
Route of administration Intravenous
[0119] Animals were divided into 11 groups, each group comprising
of eight animals. GROUP 1 received Dextrose 5% Injection, GROUP 2
received blank liposomes (before drug loading) of the present
invention, GROUP 3, GROUP 4 and GROUP 5 received Composition of
Example II at different doses, GROUP 6, GROUP 7 and GROUP 8
received CAELYX at different doses, GROUP 9, GROUP 10 and GROUP 11
received ADRIAMYCIN at different doses. The doses are provided in
Table 4.
9TABLE 4 DOSES OF DOXORUBICIN FORMULATIONS FOR REPEAT DOSE TOXICITY
STUDIES IN MICE Cumulative dose Group Dose (mg/kg No. Group (mg/kg
body weight) body weight) 1 Dextrose -- -- 2 Blank liposomes -- --
3 Composition of 1 7 4 Example II 2 14 5 3 21 6 CAELYX 1 7 7 2 14 8
3 21 9 ADRIAMYCIN 1 7 10 2 14 11 3 21
[0120] All groups received injections on alternate days, for
fourteen days via the intravenous route. The formulations were
suitably diluted with Dextrose 5% Injection before administration
to the animals. The animals were observed during the study period
of 14 days for the following:
[0121] .fwdarw.Mortality
[0122] .fwdarw.Clinical signs and symptoms
[0123] .fwdarw.Body weights
[0124] .fwdarw.Food consumption
[0125] .fwdarw.Organ weights
[0126] Results
[0127] Mortality: The percent mortality over a period of fourteen
days was recorded for all the formulations.
10TABLE 5 PERCENT MORTALITY FOR THE VARIOUS DOSES OF DOXORUBICIN
COMPOSITIONS Dose (Mg/Kg Body Group Weight) Percent Mortality
Dextrose -- 0 Blank liposomes -- 0 Composition of Example II 1 0 2
0 3 0 CAELYX 1 0 2 0 3 0 ADRIAMYCIN 1 0 2 0 3 12.5
[0128] Clinical signs: During the course of study, shedding of tail
skin and Alopecia was observed in all Doxorubicin treated groups.
Shedding of tail skin was observed in animals after five
injections. Dose dependent alopecia was observed in all of the
doxorubicin treated animals. Table 6 details the alopecia during
the course of this study.
11TABLE 6 INCIDENCE OF ALOPECIA IN MICE TREATED WITH VARIOUS
DOXORUBICIN COMPOSITIONS Formulation Grading of alopecia Dextrose
-- Blank Liposomes -- Composition of Example II (1 mg/kg) +
Composition of Example II (2 mg/kg) + Composition of Example II (3
mg/kg) ++ CAELYX (1 mg/kg) Piloerection # CAELYX (2 mg/kg) + CAELYX
(3 mg/kg) ++ ADRIAMYCIN (1 mg/kg) + ADRIAMYCIN (2 mg/kg) ++
ADRIAMYCIN (3 mg/kg) ++++ # Piloerection (raising of hair) was
observed in one out of 8 animals on day 12 of the treatment. + One
out of 8 animals showed alopecia ++ Two out of 8 animals showed
alopecia +++ Three out of 8 animals showed alopecia ++++ Four out
of 8 animals showed alopecia
[0129] Body weight: The body weight of animals were recorded on day
1, day 4, day 7 and day 14. At the dose of 2 mg/kg and 3 mg/kg, a
decrease in the body weights was observed in all drug treated
groups. The weight loss was significantly different from the
control. The body weight of animals receiving blank liposomes was
comparable to the dextrose group.
[0130] Food Consumption: From a period of 4 to 14 days Doxorubicin
treated animals showed in general a decrease in food
consumption.
[0131] Organ weights: The organs of surviving animals were
collected and weighed. The mean organ weights of all the animals
were found to be comparable in all drug treated groups.
Example IV
Evaluation of Pharmacokinetic of "Non-Pegylated Liposomal
Doxorubicin Composition of the Present Invention" With "Pegylated
Liposomal Doxorubicin Composition" (CAELYX) And "Conventional
Non-Liposomal Doxorubicin Composition" (ADRIAMYCIN) In Mice
[0132]
12 Animals used Swiss albino mice of either sex Number of groups 3
Number of animals per group 48 Animal body weight 25-30 gm Dose for
pharmacokinetic study 10 mg/kg Time points 5 min, 30 min, 1 hr, 2
hr, 5 hr, 10 hr, 15 hr, 20 hr Number of mice per time point 6 mice
Route of administration Intravenous
[0133] Blood samples after collection were centrifuged at 4000 rpm
for 20 min and the plasma was separated and frozen at -20.degree.
C. until analyzed. The frozen plasma was thawed and used for
analysis.
[0134] 1 ml of acetonitrile was added to 100 .mu.L of plasma,
vortexed for 10 mins, centrifuged at 3250 rpm for 10 mins. The
supernatant was withdrawn and 0.5 ml of saturated ZnSO.sub.4
solution was added to it. The resulting solution was vortexed for 5
mins and then centrifuged for 10 mins at 3250 rpm speed. The upper
organic layer was then withdrawn and dried under oxygen free
nitrogen gas at 60.degree. C. The residue obtained was then
reconstituted with 200 .mu.L of Solvent A containing ZnSO.sub.4.
100 .mu.L of this solution was then injected in the HPLC
column.
13 Instrument Shimadzu Liquid Chromatograph LC-10AT.sub.VP Column
C8 Thermoquest hypersil MOS (250 .times. 4.6 mm, 5 .mu.) Column
Temp Ambient Mobile Phase Solvent A: Acidified Water (pH 2.5,
adjusted with 60% Perchloric acid) & Tetrahydrofuran (80:1,
v/v) Solvent B: Acetonitrile Solvent A: Solvent B (40:60) Flow Rate
1 ml/min Detector Fluorescent Detector (RF - 10 AXL Shimadzu; Ex
460 nm and Em 550 nm Run time 15 mins
[0135] Statistical Analysis
[0136] Student's t-test was used for comparison between the three
formulations. The results are summarized in Table 1.
Example V
Comparison of Subacute Toxicity of "Non-Pegylated Liposomal
Doxorubicin Composition of the Present Invention With "Conventional
Non-Liposomal Doxorubicin Composition" (ADRIAMYCIN)" In Dogs
[0137]
14 Animals used Dogs Number of groups 3 Number of animals per group
3 Weight range of animal 10-20 kgs Dosage & administration 1
mg/kg by Intravenous infusion over 20 minutes. Administration was
done once a week (i.e. after 7 days) for 4 doses.
[0138] Pharmacological evaluation
[0139] .fwdarw.Clinical signs of toxicity
[0140] .fwdarw.Body weight
[0141] .fwdarw.Haemodynamic parameters
[0142] .fwdarw.Haematology
[0143] .fwdarw.Biochemical parameters
15TABLE 7 CLINICAL SIGNS OF TOXICITY Control (Dextrose Composition
of Signs Inj. 5%) ADRIAMYCIN Example II Dermal None of the Alopecic
lesions, erythemic None of the lesion signs were lesions seen after
third dose signs were seen Vomiting seen in this At first and
second dose - 2/3 in this group group Third and fourth dose - 1/3
Diarrhea 1/3 at after second, third and fourth dose Others
Anorexia
[0144] Body weight: ADRIAMYCIN treated groups showed decrease in
the body weight whereas Control and Composition of Example II
treated groups showed no change in body weight.
16TABLE 8 HAEMODYNAMIC PARAMETERS Control Composition (Dextrose of
Parameters Inj. 5%) ADRIAMYCIN Example II Blood pressure Normal
Normal Normal Heart rate Normal Increases by average + Normal
29.17% Respiratory rate Normal Decreases by Normal average - 42.12%
Temperature Increases body temperature during and after
administration (clinically non-significant)
[0145] Hematological parameters studied:
[0146] .fwdarw.RBC
[0147] .fwdarw.Total WBC and Differential WBC
[0148] .fwdarw.Hemoglobin
[0149] .fwdarw.Hematocrit
[0150] .fwdarw.Mean Corpuscular volume
[0151] .fwdarw.Platelet
[0152] All the above parameters studied were within normal range in
all the groups
[0153] Biochemical parameters--Increase in Creatinine phosphokinase
and lactate dehydrogenase levels were found in ADRIAMYCIN treated
groups whereas in control and the composition of Example II, there
was no significant change observed.
[0154] Liver Function Test (LFT)--Increase in Aspartate
aminotranferase, alanine aminotranferase and total bilirubin levels
were observed in ADRIAMYCIN treated groups whereas in control and
the composition of Example II no significant changes were
observed.
[0155] Kidney Function Test (KFT)--Increase in Blood Urea Nitrogen
(BUN) and creatinine were observed in ADRIAMYCIN treated groups
whereas control showed no increase. Animal group treated with
composition of Example II showed an increase in both BUN and
creatinine levels, which however, were significantly less than
ADRIAMYCIN treated groups.
Example VI
Evaluation of the Anti-Tumor Activity of "Non-Pegylated Liposomal
Doxorubicin Composition of the Present Invention" With "Pegylated
Liposomal Doxorubicin Composition" (CAELYX) Against L1210 Mouse
Leukemia And MCF-7 Human Breast Tumor Implanted In Nude Mice
[0156] Dose Preparation: Both the above doxorubicin formulations
were diluted to I1 mg/ml with sterile normal saline (0.9%).
Appropriate volumes of drug solution was administered to various
test groups on the basis of body weight so that the animals
received the drug as indicated in Tables 9 and 10.
[0157] Six week old female NCr nude (nu/nu) mice were used in both
models. The animals were housed in polycarbonate micorisolator
cages as specified in the Guide for Care and Use of Laboratory
Animals (ILAR publication, 1996, National Academy Press). The rooms
were well ventilated (greater than 10 air changes per hour) with
50% fresh air. A 12-hour light/12-hour dark photoperiod was
maintained. The room temperature was maintained between
18-26.degree. C.
[0158] The study animals were acclimatized for at least 3 days
prior to tumor inoculation.
[0159] General Description: Both liposomal formulations listed
above were tested in L1210 mouse leukemia and MCF-7 human breast
tumor models at two concentrations each against a control group
receiving saline.
[0160] L1210 Model
[0161] Tumor Cells: L1210 mouse leukemia cell line was obtained
from ATCC and propagated using standard in vitro cell expansion
methods. The cells were grown in culture media with appropriate
supplements and 10% Fetal bovine serum (FBS). The culture was then
grown in 35 T-225 flasks to 80-90% confluence. The cells were
harvested by centrifugation and the pelleted cells were resuspended
in serum-free RPMI to 10.sup.6 viable cells/ml. The animals were
injected with 0.1 ml of cell suspension using a 25 G needle.
[0162] Groups and Dosages: Each group consisted of 5 animals. Mice
were inoculated intraperitoneally with 10.sup.6 tumor cells/mouse.
Both the liposomal formulations were administered intravenously on
day 1, 5 and 9 at dosages shown in Table 9. The animals were
observed for 30 days post treatment and mortality was recorded.
17TABLE 9 Number of Total Dose/ Total Group Males/ Dose injection
number No. Females Article (mg/kg) (mg/kg) of doses 1 0/5 Saline NA
NA 3 2 0/5 CAELYX 12 4 3 3 0/5 CAELYX 6 2 3 4 0/5 Composition of 12
4 3 Example II 5 0/5 Composition of 6 2 3 Example II
[0163] The animals were examined daily and weighed twice every week
and the weights were recorded. Any mortality during the course of
the study was recorded.
[0164] The anti-tumor activity of both the liposomal formulations
were evaluated by comparing the mean survival time in each treated
group to that of the controls which received saline. The results
were expressed in terms of T/C ratios which was calculated as
follows: 1 T / C % = Mean survival time of test group Mean survival
time of control group .times. 100
[0165] A T/C.gtoreq.125% is considered significant activity.
[0166] The results of anti-tumor activity against L1210 Mouse
leukemia model are provided in Table 3. Mortalities ranged from 15
to 26 days after the first injection (Day 1). The mean survival
time of the control group, which received saline was 16.5 days.
Increase in the survival time was observed in both the drug treated
groups. Both the drug treated groups showed similar difference in
the mean survival time (T/C %) indicating that the composition of
Example II is as efficacious as CAELYX against L1210 tumor
model.
[0167] MCF-7 Model
[0168] Tumor Cells: MCF-7 human breast tumor cell line was obtained
from ATCC and propagated using standard in vitro cell expansion
methods. The cells were grown in culture media with appropriate
supplements and 10% FBS. The culture was then grown in 35 T-225
flasks to 80-90% confluence. The cells were harvested by
centrifugation and the pelleted cells were trypsinized and
resuspended in serun-free RPMI to 10.sup.7 viable cells/ml. The
animals were injected with 0.1 ml of cell suspension using a 25 G
needle.
[0169] Groups and Dosages: Each group consisted of 5 animals. Mice
were implanted with estrogen pellets 5 days prior to inoculation.
They were inoculated subcutaneously with 10.sup.7 tumor
cells/mouse. The tumor was allowed to grow until they reach a size
of 30-100 mm.sup.3. Once appropriate size has been reached
(5.sup.th day after inoculation), mice were be dosed intravenously
with the test article on day 1, 5 and 9 as shown in Table 10. Tumor
size was measured using a caliper twice weekly up to 30 days post
treatment initiation.
18TABLE 10 Number Total Total Group of Males/ Dose Dose/injection
number No. Females Article (mg/kg) (mg/kg) of doses 1 0/5 Saline --
-- -- 2 0/5 CAELYX 12 4 3 3 0/5 CAELYX 6 2 3 4 0/5 Composition 12 4
3 of Example II 5 0/5 Composition 6 2 3 of Example II
[0170] The animals were examined daily and weighed twice every week
and the weights were recorded. The length and the width for tumors
of individual mice was measured twice a week using calipers and the
approximate tumor weight (mg) from tumor dimensions (mm.times.mm)
was calculated using the formula for volume of a prolate ellipsoid:
2 L .times. W 2 2
[0171] where L is the longer of the two measurements.
[0172] The anti-tumor activity of both the liposomal formulations
were evaluated by comparing the change in tumor weight for treated
group to that of the controls, which received saline.
[0173] The change in tumor weight was calculated by subtracting the
group median tumor weight on day 5 post-inoculation of tumor cells
from group median tumor weight on the final evaluation day (day 30
post-treatment).
.upsilon.Wt=Wt.sub.final--Wt.sub.initial
[0174] The T/C ratio for all test groups was calculated as
follows:
T/C %=.upsilon.Wt Test/Wt.sub.initial of Test.times.100
[0175] A T/C 20% is considered necessary to demonstrate moderate
activity. A T/C 10% is considered significant activity.
[0176] The anti-tumor activity against MCF-7 human breast tumor
model is tabulated in Table 2.
19TABLE 11 EARLY DEATHS IN VARIOUS GROUPS OF ANIMALS Group Dosage
Mortality Control Nil 0/5 Composition of 6 mg/kg 0/5 Example II
Composition of 12 mg/kg 0/5 Example II CAELYX 6 mg/kg 2/5 CAELYX 12
mg/kg 1/5
[0177] Tumors in the control group continued to grow throughout
duration of the study reaching a maximum of 116.4 mg on the
26.sup.th day whereas tumors in the treated mice regressed
significantly during the course of the study. The tumors
disappeared completely in the group receiving 12 mg/kg of
composition of Example II formulation indicating that composition
of Example II is effective against MCF-7 human breast tumors.
[0178] Several early deaths occurred in various groups as shown in
Table 11. However, the cause of deaths seemed to be unrelated to
the tumors. There were no deaths in the saline control group, which
had the largest tumors. Some of the dead animals were necropsied,
and all of them were found to have thickened, abnormal bladders. At
the termination of the study, many of the euthanized mice, likewise
had thickened bladders. Histopathological examination of one of the
thickened bladders revealed no evidence of tumor metastasis.
Premature death of estrogenised, tumor-implanted nude mice due to
the incidence of urogenital disease.
Example VII
Determination of Maximum Tolerated Dose (MTD) And To Assess
Therapeutic Efficacy of Doxorubicin Liposomes of the Present
Invention In Nude Athymic Mice With A121 Human Ovarian Tumor
[0179] Maximum tolerated dose and assessment of therapeutic
efficacy of Doxorubicin liposomes of the present invention in nude
athymic mice with A121 human ovarian tumor was carried out in
comparison with Conventional non-liposomal formulation (ADRIAMYCIN)
and Pegylated liposomal formulation (CAELYX).
[0180] Nude athymic Ncr-nu/nu mice [4 mice/group (10 in Control
group)] were implanted subcutaneously with human A121 ovarian
tumour via trocar implant. A total of 46 animals were used in this
experiment. A total of 46 animals were utilised for the experiment.
Equivalent doses of ADRIAMYCIN, CAELYX and the composition of
Example II were evaluated intravenously. Drugs were administered
intravenously via tail vein of mice on day 5 and 12 after tumour
implant.
[0181] All treatment groups demonstrated good antitumor
efficacy.
[0182] The dosage schedule is presented below.
[0183] Dosing schedule
[0184] Control Mice: The control mice received no treatment.
[0185] ADRIAMYCIN
[0186] 12 mg/kg (6 mg/kg.times.2 inj)
[0187] 24 mg/kg (12 mg/kg.times.2 inj)
[0188] 36 mg/kg (18 mg/kg.times.2 inj)
[0189] CAELYX
[0190] 12 mg/kg (6 mg/kg.times.2 inj)
[0191] 24 mg/kg (12 mg/kg.times.2 inj)
[0192] 36 mg/kg (18 mg/kg.times.2 inj)
[0193] Composition of Example II
[0194] 12 mg/kg (6 mg/kg.times.2 inj)
[0195] 24 mg/kg (12 mg/kg.times.2 inj)
[0196] 36 mg/kg (18 mg/kg.times.2 inj)
[0197] All mice receiving the highest dosage 36 mg/kg of free drug
(18 mg/kg.times.2 ADRIAMYCIN) and 3 of 4 mice that received the
intermediate dosage of 24 mg/kg died as a result of drug toxicity.
The maximum tolerated dose (MTD) of ADRIAMYCIN is hence less than
24 mg/kg.
[0198] Mice tolerated both CAELYX and the composition of Example
II. Both the formulations were well tolerated at 36 mg/kg. However,
CAELYX appeared to cause more toxicity than the composition of
Example II and produced a greater weight loss of mice receiving the
high dose (36 mg/kg).
[0199] This study demonstrates that the composition of Example II
is better tolerated than the commercially available pegylated
liposomal preparation (CAELYX) and conventional non-liposomal
formulation (ADRIAMYCIN).
Example VIII
To Assess the Efficacy of Liposomal Doxorubicin Composition of the
Present Invention In Nude Athymic Mice Implanted With A Multidrug
Resistant, Pgp Positive, Human Colon DLD1 Tumor Xenografts
[0200] The composition of Example II along with CAELYX and
ADRIAMYCIN were subjected to efficacy studies in nude athymic mice
implanted s.c. with the drug resistant (Pgp+) DLD-1 human colon
tumor.
[0201] Animals, nude athymic mice, 4 mice/group (10 in Control
group) implanted subcutaneously with human DLD-1 colon tumor via
trocar implant.
[0202] Control: No treatment
[0203] ADRIAMYCIN
[0204] 12 mg/kg (6 mg/kg.times.2 inj)
[0205] 24 mg/kg (12 mg/kg.times.2 inj)
[0206] CAELYX
[0207] 24 mg/kg (12 mg/kg.times.2 inj)
[0208] 36 mg/kg (18 mg/kg.times.2 inj)
[0209] 48 mg/kg (24 mg/kg.times.2 inj)
[0210] Composition of Example II
[0211] 24 mg/kg (12 mg/kg.times.2 inj)
[0212] 36 mg/kg (18 mg/kg.times.2 inj)
[0213] 48 mg/kg (24 mg/kg.times.2 inj)
[0214] A total of 42 animals were utilised for the experiment.
[0215] Results
[0216] The dosages of ADRIAMYCIN were lowered to 12 and 24 mg/kg in
this study based on the toxicity observed in Example VII following
the administration of 36 mg/kg free drug. In contrast, dosages of
CAELYX and the composition of Example II were increased to 48 mg/kg
to compare their efficacies and toxicities with the free drug at
their respective MTDs. All agents were administered to nude athymic
mice i.v. via tail vein on day 5 and 12 after s.c. tumor implant
with the multidrug resistant, Pgp positive, human colon tumor
xenograft. All treatments groups demonstrated antitumor
efficacy.
[0217] However, mice receiving either of the liposomal preparations
demonstrated significantly greater antitumor efficacy. At
equivalent free drug dosages (24 mg/kg), a median tumor growth
delay of 10 days was observed with the free drug, while all mice
administered liposomal preparations had tumors that were less than
600 nm.sup.3 on day 40. No toxicity was evident at dosages of 36
mg/kg for either CAELYX or Composition of Example II.
[0218] At the highest dosages (48 mg/kg) both liposomal drug
formulations (24 mg/kg.times.2, CAELYX or Composition of Example
II), mice demonstrated >15% weight loss, and 1 of 4 animals of
each of those groups died early (day 17, 19) as a result of drug
toxicity. Therefore, the MTD of the both liposomal formulations was
similar and appeared to be less than 48 mg/kg.
[0219] In contrast to ADRIAMYCIN, the two liposomal formulations
[CAELYX--(pegylated doxorubicin) and Composition of Example II
(non-pegylated-doxorubicin)] displayed significant antitumor
efficacy against s.c. implanted, Pgp positive, multidrug resistant
human DLD1 colon tumors in nude athymic mice. At equivalent dosages
of 24 mg/kg, both liposomal formulations displayed increased
efficacy as compared with the free drug. In addition, both
liposomal formulations displayed lower toxicities as compared with
the free drug allowing more drug to be administered. The MTD for
ADRIAMYCIN appears to be about half that of the liposomal
formulations. Liposomal drug dosages of 36 mg/kg were well
tolerated.
Example IX to XIII
The Composition And Process of Example IX To XIII Are Given In
Table 12.
[0220]
20 TABLE 12 Example Example Example IX Example X XI Example XII
XIII Parameters changed Increased Particle Less Higher C14
Conventional Ingredients size Cholesterol cholesterol phospholipid
hydration DSPC 1.565 g 1.565 g 1.565 g -- 1.565 g DMPC -- -- --
1.565 g -- Cholesterol 0.521 g 0.3 g 0.74 g 0.521 g 0.521 g
Chloroform 40 ml 40 ml 40 ml 40 ml 40 ml Hydrating medium 60 ml 60
ml 60 ml 60 ml 120 ml Average particle 0.18 .mu.m 0.085 .mu.m 0.095
.mu.m 0.095 .mu.m 0.085 .mu.m Size Histidine Buffer 1.7 lt. 1.7 lt.
1.7 lt. 1.7 lt. 1.7 lt. Doxorubicin HCl 330 mg 330 mg 330 mg 330 mg
330 mg Histidine buffer 22 ml 22 ml 22 ml 22 ml 40 ml (for
solubilizing the drug) Histidine buffer 80 ml 80 ml 80 ml 80 ml --
(for dilution)
[0221] Procedure:
[0222] The Procedure of Example I was followed for Example X, XI
and XII.
[0223] In Example IX procedure of Example I was followed except for
the size reduction of liposomes which was carried out by extruding
through membranes of 0.4.mu. to 0.2.mu. to get an average size in
the range of 0.15 .mu.m to 0.25 .mu.m.
[0224] In Example XIII procedure of Example II was followed except
for the volume of hydration which was doubled.
[0225] The results of toxicological testing are given in Table
13.
21TABLE 13 Observations Example Example IX Example X Example XI
Example XII XIII Parameters changed Increased Less Higher C14
Conventional Results Particle size Cholesterol cholesterol
phospholipid hydration T.sub.1/2 in mice 2 hrs 3 hrs 5 hrs 2 hrs 4
hrs (C.sub.max and (C.sub.max and AUC not AUC not comparable)
comparable) LD.sub.50 in mice 12 mg/kg 10 mg/kg 12 mg/kg 10 mg/kg
14 mg/kg Conclusion T.sub.1/2 T.sub.1/2 C.sub.max and T.sub.1/2,
C.sub.max, Less T.sub.1/2 (with reference significantly
significantly AUC were AUC to composition less less and
significantly significantly of Example I) increased less less
toxicity
Example XIV
Liposomal Doxorubicin Composition Without Sucrose
[0226] Lipid film formation: Distearoylphosphatidylcholine (1.565
g) and cholesterol (0.521 g) were dissolved one after the other in
chloroform (40 ml) in a rotary evaporator flask. They were mixed
until a clear solution was formed. The flask was connected to a
Rotary evaporator and the water bath temperature was adjusted to
60.degree. C. The solvent was evaporated under vacuum to form thin
film of lipids on the wall of the flask. After releasing the
vacuum, the flask was rotated for approximately 5 minutes while
passing nitrogen into the flask to drive off any residual
solvent.
[0227] Hydration: The lipid film was hydrated with 60 ml of aqueous
hydration media. The aqueous hydration media was 2.04% w/v Ammonium
sulfate in water. The flask containing the lipid film and hydration
media was rotated for 30 minutes on a water bath maintained at
65-68.degree. C. to form blank liposomes.
[0228] Size reduction of blank liposomes by extrusion: The
liposomal suspension obtained from above was sized by extruding
successively through filters having pore size from 0.4 .mu.m and to
0.05 .mu.m.
[0229] Dialysis: The suspension of the sized liposomes was dialyzed
against a 0.2% w/v histidine hydrochloride solution of pH 6.5. A
tangential flow filtration system was used for the dialysis. The
dialysis was continued till extra liposomal ammonium sulfate was
removed. The absence of ammonium sulfate in extra liposomal media
was confirmed using Nesseler reagent.
[0230] Drug loading: In a round bottom flask, a 15 mg/ml solution
of Doxorubicin HCl was prepared by dissolving 216 mg of Doxorubicin
hydrochloride in 14 ml of histidine hydrochloride solution
(described above). The measured volume (40 ml) of sized and
dialyzed liposomes from above were added slowly to the round bottom
flask and mixed for one hour at 65.degree. C.
[0231] The drug loaded liposomes were treated with DOWEX to remove
the unentrapped drug.
[0232] The samples of the composition obtained before and after
treatment with DOWEX were analysed for Doxorubicin hydrochloride
content by high pressure liquid chromatography (HPLC). The results
are as follows:
22 Total Doxorubicin HCl content (before DOWEX treatment) 4.02
mg/ml Entrapped Doxorubicin HCl content (after DOWEX 4 mg/ml
treatment)
[0233] The doxorubicin hydrochloride loaded liposomes after
removing the free drug were diluted to a 2 mg/ml of doxorubicin
hydrochloride concentration using solution of histidine
hydrochloride and sucrose (described above). The liposomal
composition thus obtained was then aseptically filtered using a
sterile 0.22 .mu.m membrane filter into a sterile depyrogenated
container and was analyzed for the following parameters:
23 Appearance Red colored translucent liquid pH 6.3 Particle size
Average particle size 0.097 .mu.m Doxorubicin HCl 2.05 mg/ml
content Bacterial endotoxins Less than 2.2 EU/mg of doxorubicin
hydrochloride. Sterility Sterile
[0234] Stability studies on the compositions obtained in this
example were carried out and the observations are given in Table
14.
Example XV
Process of Making A Liposomal Doxorubicin Composition With 120mM
Ammonium Sulfate Solution
[0235] Lipid film formation: Distearoylphosphatidylcholine (1.565
g) and cholesterol (0.521 g) were dissolved one after the other in
chloroform (40 ml) in a rotary evaporator flask. They were mixed
until a clear solution was formed. The flask was connected to a
Rotary evaporator and the water bath temperature was adjusted to
60.degree. C. The solvent was evaporated under reduced pressure to
form thin film of lipids on the wall of the flask. After releasing
the vacuum, the flask was rotated for approximately 5 minutes while
passing nitrogen into the flask to drive off any residual
solvent.
[0236] Hydration: The lipid film was hydrated with 60 ml of aqueous
hydration media. The aqueous hydration media consists of Sucrose
10% w/v, Ammonium sulfate 1.58% w/v in water. The flask containing
the lipid film and hydration media was rotated for 30 minutes on a
water bath maintained at 65.degree. C.-68.degree. C. to form blank
liposomes.
[0237] Size reduction of blank liposomes by extrusion: The
liposomal suspension obtained from above was sized by extruding
successively through filters having pore size from 0.4 .mu.m and to
0.05 .mu.m.
[0238] Dialysis: The suspension of the sized liposomes was dialyzed
against a histidine buffer. A tangential flow filtration system was
used for the dialysis. The dialysis was continued till extra
liposomal ammonium sulfate was removed. The absence of ammonium
sulfate in extra liposomal media was confirmed using Nesseler
reagent. The histidine hydrochloride solution used in the dialysis
and drug loading (below) was as follows: 170.0 gm of sucrose, 3.40
gm of histidine HCl, 1.7 Liters of water, and sodium hydroxide at a
quantity sufficient to adjust pH to 6.0 to 6.5.
[0239] Drug loading: In a round bottom flask, a 15 mg/ml solution
of Doxorubicin HCl was prepared by dissolving 216 mg of Doxorubicin
hydrochloride in 14 ml of histidine hydrochloride solution
(described above). The measured volume (40 ml) of sized and
dialyzed liposomes from above were added slowly to the round bottom
flask and mixed for one hour at 65.degree. C.
[0240] The drug loaded liposomes were treated with DOWEX to remove
the unentrapped drug.
[0241] The samples of the composition obtained before and after
treatment with DOWEX were analysed for Doxorubicin hydrochloride
content by high pressure liquid chromatography (HPLC). The results
are as follows:
24 Total Doxorubicin HCl content (before DOWEX treatment) 4.11
mg/ml Entrapped Doxorubicin HCl content (after DOWEX 4.10 mg/ml
treatment)
[0242] The doxorubicin hydrochloride loaded liposomes after
removing the free drug were diluted to a 2 mg/ml of doxorubicin
hydrochloride concentration using solution of histidine
hydrochloride and sucrose (described above). The liposomal
composition thus obtained was then aseptically filtered using a
sterile 0.22 .mu.m membrane filter into a sterile depyrogenated
container and was analyzed for the following parameters:
25 Appearance Red colored translucent liquid pH 6.35 Particle size
Average particle size 0.09 .mu.m Doxorubicin HCl 2.03 mg/ml content
Bacterial endotoxins Less than 2.2 EU/mg of doxorubicin
hydrochloride. Sterility Sterile
[0243] Stability studies on the composition obtained in this
example were carried out an the observations are given in Table
14.
Example XVI
Composition of Example XIV, Example XV Along With the Composition
of Present Invention (Example II) Were Subjected For Short-Term
Stability Studies At Accelerated Temperature (25.degree. C.)
[0244] Results of doxorubicin content are given in Table 14.
26 TABLE 14 Composition of Example XV Example II Example XIV Total
Entrapped Total Entrapped Total Entrapped (mg/ (mg/ml) (mg/ml)
(mg/ml) (mg/ml) (mg/ml) ml) Initial 2.01 2.01 2.05 2.05 2.03 2.03
25.degree. C.-1 2.01 2.01 1.86 2.04 1.84 2.03 week
[0245] This example shows that the presence of sucrose is essential
for reducing leakage of encapsulated doxorubicin and ammonium
sulfate concentration in the hydration media is important. A
concentration of 120mM leads to the leakage of encapsulated
doxorubicin and hence is not satisfactory. However, the composition
of Example II containing sucrose and ammonium sulfate in a
concentration of 155mM did not leak the encapsulated doxorubicin
during the study duration.
Example XVII
Preparation of Liposomal Doxorubicin Composition By the Process of
Solvent Removal After Hydration
[0246] Distearoylphosphatidylcholine (1.565 g) and cholesterol
(0.521 g) were dissolved one after the other in ethanol (20 ml) and
pumped slowly under pressure into the aqueous hydration media which
was constantly stirred. The aqueous hydration media consisted of
Sucrose 10% w/v, Ammonium sulfate 2.04% w/v in water. This lipid
solution containing the solvent ethanol was transferred to rotary
evaporator flask. Flask was connected to a Rotary evaporator and
the water bath temperature was adjusted to 60.degree. C. Ethanol
was removed under vacuum.
[0247] Size reduction of blank liposomes by extrusion: The
liposomal suspension obtained from above was sized by extruding
successively through filters having pore size from 0.4 .mu.m and to
0.05 .mu.m.
[0248] Dialysis: The suspension of the sized liposomes was dialyzed
against a histidine buffer. A tangential flow filtration system was
used for the dialysis. The dialysis was continued till extra
liposomal ammonium sulfate was removed. The absence of ammonium
sulfate in extra liposomal media was confirmed using Nesseler
reagent. The histidine hydrochloride solution used in the dialysis
and drug loading (below) was as follows: 170.0 gm of sucrose, 3.40
gm of histidine HCl, 1.7 Liters of water, and sodium hydroxide at a
quantity sufficient to adjust pH to 6.0 to 6.5.
[0249] Drug loading: In a round bottom flask, a 15 mg/ml solution
of Doxorubicin HCl was prepared by dissolving 216 mg of Doxorubicin
hydrochloride in 14 ml of histidine hydrochloride solution
(described above). The measured volume (40 ml) of sized and
dialyzed liposomes from above were added slowly to the round bottom
flask and mixed for one hour at 65.degree. C.
[0250] The drug loaded liposomes were treated with DOWEX to remove
the unentrapped drug.
[0251] The doxorubicin hydrochloride loaded liposomes after
removing the free drug were diluted to a 2 mg/ml of doxorubicin
hydrochloride concentration using solution of histidine
hydrochloride and sucrose (described above). The liposomal
composition thus obtained was then aseptically filtered using a
sterile 0.22 .mu.m membrane filter into a sterile depyrogenated
container.
[0252] A summary of the toxicological and efficacy studies carried
out are as follows:
[0253] Example II--Non-pegylated long circulating liposomes
containing doxorubicin hydrochloride of the present invention have
shown decreased toxic effects as compared to non-liposomal
doxorubicin hydrochloride formulations (ADRIAMYCIN) and pegylated
liposomal doxorubicin hydrochloride formulations (CAELYX). The
LD.sub.50 for the non-pegylated doxorubicin liposomes of the
present invention is higher than the CAELYX and ADRIAMYCIN, thus
demonstrating that the non-pegylated doxorubicin liposomes of the
present invention have lower toxicity.
[0254] Example III--In sub-acute toxicity study, similar patter of
toxicity was observed in both the CAELYX and composition of Example
II groups whereas ADRIAMYCIN showed toxicity.
[0255] Example IV--In pharmacokinetic study, composition of Example
II and CAELYX showed comparable plasma half-life. The apparent
volume of distribution is approximately equal to the total blood
volume, which indicated low liposomal uptake by normal tissues and
was similar to CAELYX. ADRIAMYCIN showed faster clearance rate and
high volume of distribution indicating uptake of free doxorubicin
in normal tissues.
[0256] Example V--In dog toxicity study, composition of Example II
found to be better tolerated than ADRIAMYCIN.
[0257] Example VI--In tumor models of L1210 mouse leukemia and
MCF-7 human breast tumor, composition of Example II was found to be
efficacious.
[0258] Example VII--Maximum tolerated dose of the composition of
Example II was found to be much higher than ADRIAMYCIN in tumor
implanted mice.
[0259] Example VIII--Composition of Example II was found to be
efficacious in nude athymic mice implanted with a multidrug
resistant, Pgp positive, human colon DLD1 tumor xenografts.
[0260] The above Examples clearly prove that the compositions of
the present invention are very useful for reducing tumor growth.
This involves parenterally administering a therapeutically
effective amount of non-pegylated doxorubicin hydrochloride
liposomes of the present invention. The non-pegylated doxorubicin
hydrochloride liposomes have a prolonged circulation time, exhibit
decreased toxicity and do not present "Hand-Foot Syndrome" issues
and hence they are useful for reducing tumor growth.
* * * * *